1.1 Sample Information

A brief sample information is generated from the submission table for the following analysis.

Sample Index and Basic Information

Batch	Index	Data Path	region
1	1	202402271312_20240227WenhaoAAVPanNeuro500mTriFx04_VMSC05201WH	region_0
1	2	202402271312_20240227WenhaoAAVPanNeuro500mTriFx04_VMSC05201WH	region_1
1	3	202402271312_20240227WenhaoAAVPanNeuro500mTriFx04_VMSC05201WH	region_2

1.2 MERSCOPE Data Quality Summary

The summaries present the data quality assessment automatically generated by MERSCOPE for each experiment. We mainly focus on the transcripts level for each sample. So we’re looking for high density in transcripts, based on the transcripts count per field of view (FOV), transcript density in FOV, and frequency of transcripts detected.

Need to note that the low accuracy in DAPI cell boundary is not a concern, as a self-designed cell segmentation processing will take over this task.

2.1 Cell Segmentation

Based on the spatial information and images obtained from MERFISH, we developed a machine learning model using the Cellpose algorithm to distinguish individual cells via MERFISH DAPI images.

To ensure the data quality and accuracy of cells, we have defined the minimum and maximum values for cell volume and gene count per cell. The cell volume should be between [100, 1800], and the gene count per cell > 25. After filter the outliers, the qualified cells count is shown in the following table.

Outliers were filtered from the data, and the qualified cells count is presented in the following table. The identified cells after filtering are listed in the following table. In total, the study identified 219765 cells across 4 samples.

Segmented Cell Count in each Samples

Sample Index	Cell Count
region_1/	16714
region_2/	15800
region_0/	11117

2.2 Dimension Reduction

We use Scanpy for the analysis of single-cell level transcriptome data.

2.3 Cell Annotation

We use a recent published tool: Map My Cell to perform cell type annotations for each cell. It is a high resolution cell type annotator build by Alan Institude, with nested levels of classification including 34 classes and 338 subclasses.

The taxonomy is based on the Allen Mouse Brain Common Coordinate Framework version 3 (CCFv3). Part of the used abbreviations is list in the supplementary Abbreviation. Otherwise can be found in CCFv3 paper.

With the annotation, we can identify and plot the selected types of cells in each sample.

2.3.1 Cell Annotation (Low resolution class)

MapMyCell Annotation Cell Count (Low resolution class)

	class_name	count
4	01 IT-ET Glut	15093
7	02 NP-CT-L6b Glut	5284
20	30 Astro-Epen	4858
11	20 MB GABA	3352
28	19 MB Glut	2557
6	07 CTX-MGE GABA	1961
0	06 CTX-CGE GABA	1310
13	31 OPC-Oligo	1194
15	18 TH Glut	1069
10	34 Immune	1037
9	12 HY GABA	653
23	33 Vascular	626
17	24 MY Glut	552
12	27 MY GABA	522
14	21 MB Dopa	517
8	04 DG-IMN Glut	439
16	05 OB-IMN GABA	382
18	26 P GABA	352
24	23 P Glut	273
21	28 CB GABA	249
3	11 CNU-HYa GABA	245
29	14 HY Glut	238
5	13 CNU-HYa Glut	180
2	08 CNU-MGE GABA	135
19	17 MH-LH Glut	92
22	29 CB Glut	77
27	09 CNU-LGE GABA	76
25	10 LSX GABA	65
32	15 HY Gnrh1 Glut	57
26	32 OEC	53
31	03 OB-CR Glut	45
1	25 Pineal Glut	36
33	22 MB-HB Sero	28
30	16 HY MM Glut	24

2.3.2 Cell Annotation (High resolution subclass)

MapMyCell Annotation Cell Count (High resolution subclass)

	subclass_name	count
10	030 L6 CT CTX Glut	2664
6	006 L4/5 IT CTX Glut	2601
69	319 Astro-TE NN	2326
46	016 CA1-ProS Glut	2294
27	017 CA3 Glut	1872
102	318 Astro-NT NN	1568
13	022 L5 ET CTX Glut	1474
4	007 L2/3 IT CTX Glut	1178
64	020 L2/3 IT RSP Glut	1167
29	052 Pvalb Gaba	1020
14	032 L5 NP CTX Glut	909
70	005 L5 IT CTX Glut	872
39	151 TH Prkcd Grin2c Glut	870
8	053 Sst Gaba	817
125	202 PRT Tcf7l2 Gaba	762
25	004 L6 IT CTX Glut	735
24	334 Microglia NN	708
36	326 OPC NN	623
48	327 Oligo NN	571
58	321 Astroependymal NN	536
37	215 SNc-VTA-RAmb Foxa1 Dopa	517
43	046 Vip Gaba	488
94	181 IC Tfap2d Maf Glut	481
55	023 SUB-ProS Glut	480
16	028 L6b/CT ENT Glut	471
38	014 LA-BLA-BMA-PA Glut	434
17	037 DG Glut	417
21	029 L6b CTX Glut	381
159	203 LGv-SPFp-SPFm Nkx2-2 Tcf7l2 Gaba	368
128	168 SPA-SPFm-SPFp-POL-PIL-PoT Sp9 Glut	354
56	021 L4 RSP-ACA Glut	346
23	033 NP SUB Glut	345
72	197 SNr Six3 Gaba	345
142	177 SCig-an-PPT Foxb1 Glut	308
0	047 Sncg Gaba	299
9	049 Lamp5 Gaba	273
132	101 ZI Pax6 Gaba	269
20	009 L2/3 IT PIR-ENTl Glut	261
26	031 CT SUB Glut	260
31	048 RHP-COA Ndnf Gaba	250
97	213 SCsg Gabrr2 Gaba	249
15	001 CLA-EPd-CTX Car3 Glut	246
157	207 SCs Dmbx1 Gaba	194
47	338 Lymphoid NN	193
66	322 Tanycyte NN	187
18	003 L5/6 IT TPE-ENT Glut	184
35	002 IT EP-CLA Glut	178
40	045 OB-STR-CTX Inh IMN	177
12	027 L6b EPd Glut	168
11	013 COAp Grxcr2 Glut	164
134	201 PAG-RN Nkx2-2 Otx1 Gaba	161
120	333 Endo NN	158
28	198 IC Six3 En2 Gaba	155
33	010 IT AON-TT-DP Glut	149
133	331 Peri NN	149
79	204 SC Otx2 Gcnt4 Gaba	144
90	243 PGRN-PARN-MDRN Hoxb5 Glut	144
152	210 PRT Mecom Gaba	143
111	330 VLMC NN	141
65	332 SMC NN	134
50	323 Ependymal NN	128
7	019 L2/3 IT PPP Glut	127
87	199 PAG-MRN-RN Foxa2 Gaba	124
53	285 MY Lhx1 Gly-Gaba	121
22	025 CA2-FC-IG Glut	112
146	209 SCs Pax7 Nfia Gaba	109
149	155 PRC-PAG Pax6 Glut	106
184	172 PAG Pou4f1 Ebf2 Glut	105
80	304 NTS-PARN Neurod2 Gly-Gaba	100
119	195 SNr-VTA Pax5 Npas1 Gaba	97
84	119 SI-MA-LPO-LHA Skor1 Glut	97
91	042 OB-out Frmd7 Gaba	97
176	167 PRC-PAG Tcf7l2 Irx2 Glut	96
101	200 PAG-ND-PCG Onecut1 Gaba	94
141	163 APN C1ql2 Glut	93
124	212 SCs Lef1 Gli3 Gaba	92
165	166 MRN Pou3f1 C1ql4 Glut	92
96	050 Lamp5 Lhx6 Gaba	91
61	034 NP PPP Glut	86
77	214 IPN Otp Crisp1 Gaba	84
274	190 ND-INC Foxd2 Glut	83
117	154 PF Fzd5 Glut	81
51	300 PARN-MDRNd-NTS Gbx2 Gly-Gaba	78
185	174 PAG Pou4f2 Mesi2 Glut	77
86	254 VCO Mafa Meis2 Glut	77
151	308 DCO Il22 Gly-Gaba	77
153	170 PAG-MRN Tfap2b Glut	76
174	187 SCsg Pde5a Glut	74
54	309 CB PLI Gly-Gaba	73
137	165 PAG-MRN Pou3f1 Glut	67
129	164 APN C1ql4 Glut	67
195	089 PVR Six3 Sox3 Gaba	62
41	018 L2 IT PPP-APr Glut	62
178	277 DTN-LDT-IPN Otp Pax3 Gaba	60
93	057 NDB-SI-MA-STRv Lhx8 Gaba	58
181	206 SCm-PAG Cdh23 Gaba	58
189	325 CHOR NN	58
60	336 Monocytes NN	58
167	142 HY Gnrh1 Glut	57
224	173 PAG Pou4f2 Glut	56
156	176 SCig Foxb1 Glut	55
114	141 PH-SUM Foxa1 Glut	55
123	238 NTS Phox2b Glut	55
106	335 BAM NN	54
67	283 PRP Otp Gly-Gaba	54
71	314 CB Granule Glut	54
113	086 MPO-ADP Lhx8 Gaba	53
88	328 OEC NN	53
45	145 MH Tac2 Glut	53
136	169 PAG-SC Pou4f1 Zic1 Glut	52
205	217 PB Lmx1a Glut	51
138	161 PAG Pou4f3 Glut	50
208	102 DMH-LHA Gsx1 Gaba	48
107	222 PB Evx2 Glut	48
168	205 SC-PAG Lef1 Emx2 Gaba	48
49	264 PRNc Otp Gly-Gaba	48
42	237 PRP-NI-PRNc-GRN Otp Glut	47
273	231 IPN-LDT Vsx2 Nkx6-1 Glut	46
92	015 ENTmv-PA-COAp Glut	45
95	263 CS-RPO Meis2 Gaba	44
170	329 ABC NN	44
270	159 IF-RL-CLI-PAG Foxa1 Glut	44
2	056 Sst Chodl Gaba	42
183	180 SCiw Pitx2 Glut	42
243	251 NTS Dbh Glut	41
73	093 RT-ZI Gnb3 Gaba	40
112	098 AHN-SBPV-PVHd Pdrm12 Gaba	40
143	146 LH Pou4f1 Sox1 Glut	39
135	039 OB Meis2 Thsd7b Gaba	38
30	008 L2/3 IT ENT Glut	37
163	228 PSV Pvalb Lhx2 Glut	37
116	147 AD Serpinb7 Glut	37
148	171 PAG Pou4f1 Bnc2 Glut	37
211	211 SC Tnnt1 Gli3 Gaba	36
150	248 MV-SPIV Zic4 Neurod2 Glut	36
1	262 Pineal Crx Glut	36
59	044 OB Dopa-Gaba	36
103	012 MEA Slc17a7 Glut	35
98	311 CBX MLI Megf11 Gaba	35
200	149 PVT-PT Ntrk1 Glut	35
44	282 POR Spp1 Gly-Gaba	35
206	134 PH-ant-LHA Otp Bsx Glut	35
155	246 CU-ECU-SPVI Foxb1 Glut	35
186	312 CBX MLI Cdh22 Gaba	34
63	011 L2 IT ENT-po Glut	33
182	051 Pvalb chandelier Gaba	33
204	247 MV-SPIV Phox2b Ebf3 Lbx1 Glut	33
32	306 SPVI-SPVC Sall3 Lhx1 Gly-Gaba	32
237	097 PVHd-SBPV Six3 Prox1 Gaba	31
199	152 RE-Xi Nox4 Glut	30
52	094 SCH Six6 Cdc14a Gaba	30
223	085 SI-MPO-LPO Lhx8 Gaba	30
108	041 OB-in Frmd7 Gaba	28
179	216 MB-MY Tph2 Glut-Sero	28
110	208 SC Lef1 Otx2 Gaba	28
169	250 CBN Neurod2 Pvalb Glut	27
252	271 NI-RPO Gata3 Nr4a2 Gaba	27
130	279 PSV Pax2 Gly-Gaba	27
177	175 SC Bnc2 Glut	27
260	133 PVH-SO-PVa Otp Glut	25
140	106 PVpo-VMPO-MPN Hmx2 Gaba	25
221	337 DC NN	24
218	035 OB Eomes Ms4a15 Glut	24
162	224 PCG-PRNr Vsx2 Nkx6-1 Glut	24
209	100 AHN Onecut3 Gaba	24
173	287 MV-SPIV-PRP Dmbx1 Gly-Gaba	24
212	109 LGv-ZI Otx2 Gaba	24
164	108 ARH-PVp Tbx3 Gaba	23
62	315 DCO UBC Glut	23
131	313 CBX Purkinje Gaba	23
257	104 TU-ARH Otp Six6 Gaba	23
188	316 Bergmann NN	22
76	038 DG-PIR Ex IMN	22
201	070 LSX Prdm12 Slit2 Gaba	22
158	036 HPF CR Glut	21
81	270 LDT-DTN Gata3 Nfix Gaba	21
175	105 TMd-DMH Foxd2 Gaba	20
126	061 STR D1 Gaba	20
68	114 COAa-PAA-MEA Barhl2 Glut	20
127	156 MB-ant-ve Dmrta2 Glut	19
220	188 SCop Sln Glut	19
139	115 MS-SF Bsx Glut	18
109	317 Astro-CB NN	18
264	305 SPVI-SPVC Sall3 Nfib Gly-Gaba	17
232	196 PAG-PPN Pax5 Sox21 Gaba	17
241	103 PVHd-DMH Lhx6 Gaba	17
233	124 MPN-MPO-PVpo Hmx2 Glut	17
210	160 PAG-SC Neurod2 Meis2 Glut	17
242	132 AHN-RCH-LHA Otp Fezf1 Glut	16
238	268 CS-PRNr-DR En1 Sox2 Gaba	16
171	144 MM Foxb1 Glut	16
191	255 SPVO Mafa Meis2 Glut	16
105	192 PPN-CUN-PCG Otp En1 Gaba	16
118	066 NDB-SI-ant Prdm12 Gaba	16
239	320 Astro-OLF NN	15
160	221 LDT-PCG Vsx2 Lhx4 Glut	15
57	054 STR Prox1 Lhx6 Gaba	14
154	278 NLL Gata3 Gly-Gaba	14
144	139 PH-LHA Foxb1 Glut	14
74	225 PRNc-NI-SG-RPO Vsx2 Nr4a2 Glut	13
213	267 CS-PRNr-PCG Tmem163 Otp Gaba	13
166	117 LHA Barhl2 Glut	13
190	069 LSX Nkx2-1 Gaba	13
254	058 PAL-STR Gaba-Chol	13
180	062 STR D2 Gaba	13
145	083 CEA-BST Rai14 Pdyn Crh Gaba	12
161	296 RPA Pax6 Hoxb5 Gly-Gaba	12
266	107 DMH Hmx2 Gaba	12
34	191 PAG-MRN Rln3 Gaba	12
271	138 PH Pitx2 Glut	12
287	060 OT D3 Folh1 Gaba	12
300	153 MG-POL-SGN Nts Glut	12
236	194 MRN-VTN-PPN Pax5 Cdh23 Gaba	12
249	118 ADP-MPO Trp73 Glut	12
89	065 IA Mgp Gaba	11
258	129 VMH Nr5a1 Glut	11
194	293 PAS-MV Ebf2 Gly-Gaba	11
235	265 PB Sst Gly-Gaba	11
229	140 PMd-LHA Foxb1 Glut	10
3	076 MEA-BST Lhx6 Nfib Gaba	10
245	071 LSX Prdm12 Zeb2 Gaba	10
99	235 PG-TRN-LRN Fat2 Glut	10
225	082 CEA-BST Ebf1 Pdyn Gaba	10
309	186 SCop Pou4f2 Neurod2 Glut	10
255	080 CEA-AAA-BST Six3 Sp9 Gaba	9
248	260 MDRNv Crp Glut	9
247	182 CUN-PPN Evx2 Meis2 Glut	9
240	297 CU-ECU Pax2 Gly-Gaba	9
196	095 DMH Prdm13 Gaba	9
222	272 LDT-PCG-CS Gata3 Lhx1 Gaba	9
214	307 RO-RPA Pkd2l1 Gaba	9
147	162 CUN Evx2 Lhx2 Glut	9
198	290 MY Prox1 Lmo7 Gly-Gaba	9
227	126 ARH-PVp Tbx3 Glut	9
202	276 LDT-PCG St18 Gaba	9
121	143 MM-ant Foxb1 Glut	8
265	055 STR Lhx8 Gaba	8
82	073 MEA-BST Sox6 Gaba	8
187	090 BST-MPN Six3 Nrgn Gaba	8
253	302 MV Xdh Gly-Gaba	8
250	245 SPVI-SPVC Tlx3 Ebf3 Glut	8
192	092 TMv-PMv Tbx3 Hist-Gaba	8
268	059 GPe-SI Sox6 Cyp26b1 Gaba	7
19	099 SBPV-PVa Six6 Satb2 Gaba	7
286	234 MEV Ppp1r1c Glut	7
311	179 SCdg-PAG Tfap2b Glut	7
294	064 STR-PAL Chst9 Gaba	7
207	068 LSX Otx2 Gaba	7
301	157 RN Spp1 Glut	7
85	072 LSX Sall3 Lmo1 Gaba	7
231	310 CBX Golgi Gly-Gaba	7
244	116 AVPV-MEPO-SFO Tbr1 Glut	7
269	026 NLOT Rho Glut	6
277	067 LSX Sall3 Pax6 Gaba	6
217	233 NLL-SOC Spp1 Glut	6
104	063 STR D1 Sema5a Gaba	6
122	284 GRN-IRN-MDRNd Ikzf1 Gly-Gaba	6
197	273 PDTg-PCG Pax6 Gaba	6
296	131 LHA-AHN-PVH Otp Trh Glut	6
261	075 MEA-BST Lhx6 Nr2e1 Gaba	6
272	135 STN-PSTN Pitx2 Glut	6
267	291 NTS-MDRNd Prox1 Zic1 Gly-Gaba	5
78	298 PRP Gata3 Slc6a5 Gly-Gaba	5
293	088 BST Tac2 Gaba	5
306	295 CBN Dmbx1 Gaba	5
297	226 PRNc-PARN Tlx1 Glut	5
5	113 MEA-COA-BMA Ccdc42 Glut	5
298	256 SPVC Mafa Glut	5
230	220 PB Pax5 Glut	5
100	127 DMH-LHA Vgll2 Glut	5
307	185 SCig Tfap2b Chrnb3 Glut	4
281	239 MARN-GRN Pyy Glut	4
282	130 LHA Pmch Glut	4
288	087 MPN-MPO-LPO Lhx6 Zfhx3 Gaba	4
295	193 MRN-PPN-CUN Pax8 Gaba	4
303	150 CM-IAD-CL-PCN Sema5b Glut	4
280	125 DMH Hmx2 Glut	4
203	257 SPVC Ccdc172 Glut	4
234	286 PPY-PGRNl Vip Glyc-Gaba	4
83	281 POR Gata3 Gly-Gaba	4
193	299 MARN-PPY Ngfr Gly-Gaba	4
263	294 MV Pax6 Gly-Gaba	4
262	040 OB Trdn Gaba	4
259	074 MEA-BST Lhx6 Sp9 Gaba	4
215	136 PMv-TMv Pitx2 Glut	4
75	223 B-PB Nr4a2 Glut	4
278	232 LDT Vsx2 Nkx6-1 Nfib Glut	3
279	123 DMH Nkx2-4 Glut	3
285	236 IRN Vip Glut	3
246	091 ARH-PVi Six6 Dopa-Gaba	3
304	280 NLL-po Pax7 Gaba	3
315	112 GPi Tbr1 Cngb3 Gaba-Glut	3
292	122 LHA-MEA Otp Glut	3
216	274 PDTg Otp Shroom3 Gaba	3
319	189 PAG Ucn Glut	2
305	288 MDRN Hoxb5 Ebf2 Gly-Gaba	2
317	240 MDRNv Lhx4 Qrfprl Glut	2
312	178 SCig Foxb1 Otx2 Glut	2
316	184 PAG Tcf24 Glut	2
228	230 PRNr Otp Nfib Glut	2
320	081 ACB-BST-FS D1 Gaba	2
299	043 OB-mi Frmd7 Gaba	2
276	261 HB Calcb Chol	2
226	084 BST-SI-AAA Six3 Slc22a3 Gaba	2
115	292 MV Nkx6-1 Gly-Gaba	2
251	078 SI-MA-ACB Ebf1 Bnc2 Gaba	2
256	120 MEA Otp Foxp2 Glut	2
172	079 CEA-BST Six3 Cyp26b1 Gaba	2
283	219 PB-SUT Tlx3 Lhx2 Glut	2
289	137 PH-an Pitx2 Glut	2
308	258 SPVC Nmu Glut	1
310	218 PSV Lmx1a Trpv6 Glut	1
302	269 LDT Fgf7 Gaba	1
284	275 PDTg Otp Olig3 Gaba	1
313	301 MV Nr4a2 Gly-Gaba	1
314	244 MV-SPIV Slc6a2 Glut	1
219	229 PB-NTS Phox2b Ebf3 Lmx1b Glut	1
290	252 DMX VII Tbx20 Chol	1
318	024 L5 PPP Glut	1
291	183 PBG Mtnr1a Glut-Chol	1
275	242 PGRNd Dmbx1 Glut	1

2.3.3 Spatial cell type (Neurons)

2.3.4 Spatial cell type (Non-Neurons)

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Cell types & Regions

Astro, Astrocyte;

ABC, arachnoid barrier cells;

BAM, border-associated macrophages;

BLA, Basolateral amygdala;

CB, cerebellum;

CGE, caudal ganglionic eminence;

CHOR, choroid plexus;

CNU, cerebral nuclei;

CR, Cajal–Retzius;

CT, corticothalamic;

CTX, cerebral cortex;

CTXsp, cortical subplate;

DC, dendritic cells;

DCO, dorsal cochlear nucleus;

DG, dentate gyrus;

EA, extended amygdala;

Endo, endothelial cells;

ENT, Entorhinal area;

ENTl, Entorhinal area, lateral part;

Epen, ependymal;

EPI, epithalamus;

ET, extratelencephalic;

GC, granule cell;

HB, hindbrain;

HPF, hippocampal formation;

HY, hypothalamus;

HYa, anterior hypothalamic;

IMN, immature neurons;

IT, intratelencephalic;

L6b, layer 6b;

LGE, lateral ganglionic eminence;

LH, lateral habenula;

LSX, lateral septal complex;

MB, midbrain;

MGE, medial ganglionic eminence;

MH, medial habenula;

MM, medial mammillary nucleus;

MY, medulla;

NN, non-neuronal;

NP, near-projecting;

NT, non-telencephalon;

OB, olfactory bulb;

OEC, olfactory ensheathing cells;

OLF, olfactory areas;

Oligo, oligodendrocytes;

OPC, oligodendrocyte precursor cells;

P, pons;

PAL, pallidum;

Peri, pericytes;

PIR, piriform cortex;

SMC, smooth muscle cells;

STR, striatum;

TE, telencephalon;

TH, thalamus;

UBC, unipolar brush cells;

VLMC, vascular leptomeningeal cells.

Neurotransmitter types

Chol, cholinergic;

Dopa, dopaminergic;

GABA, GABAergic;

Glut, glutamatergic;

Glyc, glycinergic;

Hist, histaminergic;

Nora, noradrenergic;

Sero, serotonergic